Hawkins J, Marcy A
Department of High Throughput Screening and Automation, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
Protein Expr Purif. 2001 Jul;22(2):211-9. doi: 10.1006/prep.2001.1447.
Itk is a Tec family tyrosine kinase found in T cells that is activated upon ligation of the T cell receptor (TCR/CD3), CD2, or CD28. Itk contains five domains in addition to the catalytic domain: pleckstrin homology, Tec homology which contains a proline-rich region, Src homology 3, and Src homology 2. To provide a basis for understanding the contribution of these various domains to catalysis, recombinant Itk was purified and its substrate specificity determined by steady-state kinetic methods. Measurements of the rates of phosphorylation of various protein substrates, including Src associated in mitosis 68K protein (SAM68), CD28, linker for activation of T cells, and CD3 zeta, at a fixed concentration indicated that SAM68 was phosphorylated most rapidly. Wild-type Itk and three Itk mutants were characterized by comparing their activity (k(cat)) using the SAM68 substrate. A deletion mutant removing the pleckstrin homology domain and part of the Tec homology domain (Itk(Delta152)) had approximately 10-fold less activity than wild type, a mutant with an altered proline-rich domain (P158A,P159A) had a more dramatic 100-fold loss of activity, and the catalytic domain alone was essentially inactive. Itk(Delta152) had K(m) values for ATP and SAM68 nearly identical to those of the wild-type enzyme, while Itk(P158A,P159A) had approximately 3-fold higher K(m) values for each substrate. SAM68 phosphorylation by the wild-type and mutant enzymes in the presence of several tyrosine kinase inhibitors were compared using a homogeneous time-resolved fluorescence assay. Both the Itk(Delta152) deletion mutant and the Itk(P158A,P159A) mutant had IC(50) values similar to those of the wild-type enzyme for staurosporine, PP1, and damnacanthal. These comparisons, taken together with the similar K(m) values for ATP and SAM68 substrate between the wild-type and the mutant enzymes, indicate that the amino acids in the N-terminal 152 residues and proline-rich domains enhance catalysis by affecting turnover rate rather than substrate binding.
Itk是一种在T细胞中发现的Tec家族酪氨酸激酶，在T细胞受体（TCR/CD3）、CD2或CD28连接后被激活。除催化结构域外，Itk还包含五个结构域：普列克底物蛋白同源结构域、含有富含脯氨酸区域的Tec同源结构域、Src同源结构域3和Src同源结构域2。为了理解这些不同结构域对催化作用的贡献，纯化了重组Itk，并通过稳态动力学方法确定其底物特异性。在固定浓度下测量各种蛋白质底物（包括有丝分裂相关的68K Src结合蛋白（SAM68）、CD28、T细胞激活连接蛋白和CD3 ζ）的磷酸化速率，结果表明SAM68的磷酸化速度最快。通过使用SAM68底物比较野生型Itk和三个Itk突变体的活性（k(cat)）来对它们进行表征。缺失普列克底物蛋白同源结构域和部分Tec同源结构域的缺失突变体（Itk(Delta152)）的活性比野生型低约10倍，富含脯氨酸结构域发生改变的突变体（P158A、P159A）活性急剧丧失100倍，而单独的催化结构域基本无活性。Itk(Delta152)对ATP和SAM68的K(m)值与野生型酶几乎相同，而Itk(P158A、P159A)对每种底物的K(m)值大约高3倍。使用均相时间分辨荧光测定法比较了野生型和突变型酶在几种酪氨酸激酶抑制剂存在下对SAM68的磷酸化作用。对于星形孢菌素、PP1和金丝桃素，Itk(Delta152)缺失突变体和Itk(P158A、P159A)突变体的IC(50)值与野生型酶相似。这些比较，连同野生型和突变型酶之间ATP和SAM68底物的K(m)值相似，表明N端152个残基和富含脯氨酸结构域中的氨基酸通过影响周转速率而非底物结合来增强催化作用。
